Fungal infections by members of the genus Fusarium can manifest as superficial skin lesions, however in immunocompromised individuals these fungi are capable of causing a life-threatening disease such as fusariosis where the fungus is invasive and often disseminates throughout the patient. Further complicating treatment of severe infections is the intrinsic resistance of these fungi to many of the clinically used antifungal agents such as the azoles and echinocandins. Two groups of fungi within this genus are frequently responsible for causing the most severe form of the disease, Fusarium solani and Fusarium oxysporum. Recently, both of these fungi have been shown to have small supernumerary chromosomes that are absent in other genera of Fusarium not commonly associated with human infections. Previously, I have developed the use of the larvae of the greater wax moth Galleria mellonella as an alternative host to study Fusarium pathogenesis. These studies revealed that thermotolerance is a major factor for the ability of Fusarium to infect humans. As an independent investigator, I seek to identify the molecular mechanisms that enable Fusarium spp. to cause disease in humans. The presence of the small chromosomes within the species most frequently associated with clinical infections suggests these chromosomes may carry genes involved in establishing disease in humans. Small chromosomes that are frequently found in clinical F. solani and F. oxysporum isolates will be identified by pulse-field gel electrophoresis. These chromosomes will then be sequenced in conjunction with the entire genome of five representative isolates. Genes on the small chromosomes that have characteristics of potential virulence factors (similarity to known virulence genes in other fungi, transcription factors, proteins with secretion signals, etc.) will e individually mutated and assessed to determine their role in virulence. Additionally, other traits will be analyzed such as antifungal resistance and biofilm formation. Genes involved in sensing and establishing disease in mammals will also be identified using a transcriptome analysis approach. Genes significantly induced upon sensing and initial colonization of pulmonary tissue will be identified by RNA-seq. Highly expressed fungal genes will then be mutated and characterized for traits involved in pathogenicity. Research conducted during this funding period will provide information necessary for a subsequent a R01 application. The intrinsic resistance of Fusarium spp. to conventional antifungal therapies has made it necessary to identify other potential antifungal targets for successful treatment of this emerging pathogen. Research detailed within this proposal will identify genes involved in the virulence of the fungus or other traits which may potentially be exploited as antifungal target sites. Further understanding of the development of the disease may yield additional treatment strategies to overcome these fungi that currently have an unacceptably high mortality rate.